This invention relates to the field of genetic engineering, and more particularly to transformation of plants with heterologous fatty acid desaturase genes modified for optimum expression in plants.
Several publications are referenced in this application in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.
Alteration of fatty acid desaturation in plants is of interest to plant biologists and food scientists alike, due to the influence of unsaturated fatty acids on the health benefits and flavors of foods, as well as the role of these molecules in plant biological processes. For a nation interested in healthy diet, the quality of fats and oils depends on their fatty acid composition, with oils high in monounsaturated fatty acids (e.g., canola, olive) gaining popularity as new health benefits are discovered. Considering the flavors of plant foods, many flavor-producing compounds are derived from peroxidation of unsaturated fatty acids. Thus, efforts are being made to produce plants with increased amounts of unsaturated fatty acids, preferably monounsaturated fatty acids.
In animal and fungal cells, monounsaturated fatty acids are aerobically synthesized from saturated fatty acids by a microsomal xcex94-9 fatty acid desaturase that is membrane bound and cytochrome b5-dependent. A double bond is inserted between the 9- and 10-carbons of palmitoyl (16:0) and stearoyl (18:0) CoA to form palmitoleic (16:1) and oleic (18:1) acids. In the reaction mechanism, electrons are transferred from NADH-dependent cytochrome b5 reductase, via the heme-containing cytochrome b5 (Cyt b5) molecule, to the xcex94-9 fatty acid desaturase. The major form of cytochrome b5 in animal, fungal and plant cells exists as an independent protein molecule that is anchored to the membrane by a short, carboxyl terminal, hydrophobic stretch of amino acids. The carboxyl terminal anchor orients the heme group of the Cyt b5 on the membrane surface and allows it to translationally diffuse across the surface of the membrane. This property of lateral mobility allows this form of cytochrome b5 to participate as an electron donor to a number of different proteins that catalyze a variety metabolic reactions on the membrane surface, including fatty acid desaturases, various sterol biosynthetic enzymes and a variety of cytochrome P450 mediated reactions. While this contributes to the versatility of Cyt b5 as an electron donor, it also implies that the major form of cytochrome b5 shuttles between its redox partners by translational diffusion across the surface of the membrane (Strittmatter and Rogers, Proc. Natl. Acad. Sci. USA, 72: 2658-2661, (1975; Lederer, Biochimie 76: 674-692, 1994). Furthermore, this mechanism suggests that an independent, membrane bound cytochrome b5 molecule can potentially limit the rate of the metabolic reaction, depending on its abundance, its location on the membrane surface, its proximity to the electron acceptor, and the rate at which it can move and orient itself to the acceptor on the membrane surface.
In plants, unsaturated fatty acids are formed and incorporated into complex lipids in two distinct cellular compartments. De novo fatty acid synthesis occurs almost exclusively in the plastids, producing the saturated species 16:0-ACP (acyl carrier protein) and 18:0-ACP. 18:1-ACP is formed from 18:0-ACP in the plastid by a soluble, ferredoxin-dependent xcex94-9 desaturase. These fatty acids are then shunted into one of two routesxe2x80x94a plastid-localized xe2x80x9cprocaryoticxe2x80x9d pathway or a cytosolic/ER (endoplasmic reticulum) xe2x80x9ceucaryoticxe2x80x9d pathwayxe2x80x94for further modification and acylation into glycerolipids (Somerville and Browse, Science 252: 80-87, 1991). The acyl ACPs that are shunted into the prokaryotic pathway remain within the plastid and are used for the synthesis of phosphatidic acid and further conversion to chloroplast glycerolipids. The fatty acyl groups of those lipids may be further desaturated by plastid desaturases that also use ferrodoxin as the electron donor.
Acyl-ACPs that are shunted into the eukaryotic pathway are converted to free fatty acids, transported across the chloroplast membrane into the cytoplasm where they are converted to acyl CoA thioesters by acyl CoA synthetase. Those fatty acids are then converted to cytoplasmic/ER phosphatidic acid which can then be converted to membrane glycerophospholipids, or storage lipids, in the form of triacylglycerols and sterol esters that are the major components of plant oils.
Most polyunsaturated 18-carbon plant fatty acids appear to be formed in the cytosol by the ER-bound desaturases (Table 1). Once the 18:1 fatty acid is incorporated into phospholipid, an ER-bound desaturase can catalyze the formation of a xcex94-12 double bond in the fatty acyl chain to form xcex94-9,12 18:2. Other ER bound desaturase enzymes can act on 18:2 to introduce a xcex94-15 double bond to form xcex949,12,15 18:3. These desaturase are thought to be similar to animal and fungal desaturases because they are membrane bound and appear to require a cytochrome b5-mediated electron transport chain.
The conversion of saturated fatty acyl chains to monounsaturated species in plants appears to be confined to the chloroplasts. No xcex94-9 desaturase activity has been identified in the cytoplasm or endoplasmic reticulum of plants. The soluble plant chloroplast xcex94-9 desaturase is highly specific for 18:0-ACP as a substrate and does not desaturate 16:0-ACP (Somerville and Browse, supra). As a result, only a small amount of 16:1 is present in most higher plants, while the pool of 16:0 is concomitantly larger due to its disfavor as a substrate for the plant desaturase. By comparison, a larger amount of 18:1 is found in higher plant cells, with a correspondingly lesser amount of 18:0. Thus, for the purpose of increasing the concentration of mono-unsaturated lipids in a plant, the 16:0 fatty acid constitutes a significant pool of available substrate that is under-utilized by the endogenous plant desaturase.
In contrast to the plant xcex94-9 desaturase, fungal and animal xcex94-9 desaturases efficiently convert a wide range of saturated fatty acids with differing hydrocarbon chain lengths to monounsaturated fatty acids. The Saccharomyces cerevisiae enyzme, for example, efficiently desaturates even and odd chain fatty acyl CoA substrates from 13 carbons to 19 carbons in length. A broad functional homology exists among various Cyt b5-dependent desaturases, as evidenced, for example, by the successful expression of the rat xcex94-9 desaturase in yeast (Stukey et al., J. Biol. Chem. 2: 20144-20149, 1990).
The rat and yeast xcex94-9 desaturase genes have been expressed in plants: both the rat and the yeast genes have been expressed in tobacco (Grayburn et al., BioTechnology 10: 675-678, 1992 (rat); Polashock et al., Plant Physiol. 100: 894-901, 1992 (yeast), and the yeast gene has also been expressed in tomato (Wang et al., J. Agric. Food Chem. 44: 3399-3402, 1996). The yeast xcex94-9 desaturase has been shown to function in tobacco and tomato, leading to increases in the level of monounsaturated fatty acids (both 16:1 and 18:1) and other compounds derived from monounsaturated fatty acids (e.g., polyunsaturated fatty acids, hexanal, 1-hexanol, heptanal, trans-2-octenal) (Polashock et al., supra; Wang et al; supra) . Expression of the rat desaturase also led to an increase in monounsaturated 16- and 18-carbon fatty acids (Grayburn et al., supra).
From the foregoing, it can be seen that transgenic plants expressing animal or fungal xcex94-9 desaturase genes can be improved in their unsaturated fatty acid composition by virtue of the activity of the foreign enzyme. Of further advantage, it has recently been discovered that some fungal xcex94-9 desaturases (e.g., Saccharomyces cerevisiae) are fusion proteins comprising an intrinsic Cyt b5 domain (Mitchell and Martin, J. Biol. Chem. 270: 29766-29772, 1995). When this gene is expressed, sufficient Cyt b5 is produced to drive the desaturase reaction at an optimum level and is not dependent on existing plant Cyt b5. The known animal xcex94-9 desaturases do not contain this fused Cyt b5 motif and must rely on independently-produced Cyt b5 to provide the electrons for the reactions.
Though fungal or animal xcex94-9 desaturases (e.g. the S. cerevisiae desaturase or the animal desaturases) may be expressed and functional in certain plants, their expression is likely less than optimal in plants, and expression may not even be possible in other plant species, due to several factors, including differences in codon usage and codon preference in plants as compared to fungi, and among different plant species and the presence of cryptic intron splicing signals, among others. All of these factors can lead to poor expression, or no expression, of a non-plant foreign gene in a plant cell.
Accordingly, in order to make use of non-plant fatty acid desaturases, particularly those such as the S. cerevisiae xcex94-9 desaturase comprising an internal Cyt b5 motif, a need exists to design modified desaturase-encoding DNA molecules that are customized for expression in plant cells and specific plant tissues. It would be of even greater advantage to optimize such modified DNA molecules for expression in particular plant species, such as those that are grown and harvested primarily for oils.
According to one aspect of the invention, a synthetic fatty acid desaturase gene for expression in a multi-cellular plant is provided, the gene comprising a desaturase domain and a Cyt b5 domain, wherein the gene is customized for expression in a plant cytoplasm. In one embodiment, the synthetic gene is customized for expression in a monocotyledonous plant. In another embodiment, the synthetic gene is customized for expression in a dicotyledonous plant. In a preferred embodiment, the synthetic gene is customized for expression in a plant genus selected from the group consisting of Arabidopsis, Brassica, Phaeseolus, Oryza, Olea, Elaeis (Oil Palm) and Zea.
In a preferred embodiment of the invention, the desaturase is a cytosolic xcex94-9 desaturase. The Saccharomyces cerevisiae xcex94-9 desaturase is particularly preferred.
In another embodiment of the invention, the synthetic gene is customized from a naturally occurring gene comprising both a desaturase domain and a cyt b5 domain. Alternatively, the synthetic gene is a chimeric gene comprising a desaturase domain and a heterologous cyt b5 domain.
In another embodiment, the synthetic gene is customized from a naturally occurring gene such that the synthetic gene and the naturally occurring gene encode an identical amino acid sequence. Alternatively, the synthetic gene is customized from a naturally occurring gene such that the synthetic gene and the naturally occurring gene encode a similar and functionally conserved amino acid sequence.
In another embodiment, a naturally occurring or a synthetic gene is customized so that specific amino acid modification are made to enhance the function of the encoded protein. Examples of such modifications include changing amino acids that are subjected to phosphorylation or other post-translational modifications that may alter or regulate the activity of the xcex94-9 desaturase enzyme.
In another embodiment of the invention, elements of a naturally occurring or a synthetic desaturase gene that are not essential for enzymatic function are replaced or linked with elements derived from plant ER lipid biosynthetic genes that are normally expressed in maturing seeds or other plant tissues. The improved expression of the modified gene produced by the inclusion or substitution of plant DNA sequences in the synthetic gene will result from native plant signal or control elements in those sequences that affect desaturase gene expression at one or more levels.
According to another aspect of the invention, a method is provided for constructing and customizing a bifunctional desaturase/cyt b5 encoding gene for expression in the cytosol of a multicellular plant. The method comprises (a) providing a DNA molecule comprising a desaturase-encoding moiety operably linked to a cyt b5-encoding moiety, said DNA molecule producing the bifunctional polypeptide in a non-customized form; (b) back-translating the polypeptide sequence using preferred codons for expression in a multicellular plant, thereby producing a back-translated nucleotide sequence; (c) analyzing the back-translated nucleotide sequence for features that could diminish or prevent expression in the plant cytoplasm, including, optionally (1) probable intron splice sites (characterized by T-rich regions); (2) plant polyadenylation signals (e.g., AATAAA); (3) polymerase II termination sequence (e.g., CAN(7-9)AGTNNAA, where N is any nucleotide); (4) hairpin consensus sequences (e.g., UCUUCGG); and (5) the sequence-destabilizing motif ATTTA; (d) modifying the analyzed sequence to correct or remove the features that could diminish or prevent expression in the plant cytoplasm; and, optionally, (e) introducing desirable cloning features, such as restriction sites, into the sequence in a manner that does not materially affect the desired codon usage or final polypeptide sequence.
The method set forth above may be adapted by incorporating into the customized gene one or more genomic segments from plant desaturase or other ER lipid biosynthetic genes, which are determined to further optimize gene expression in plants. This method comprises (1) identifying cDNA sequences that have potential to comprise such beneficial elements, (2) creating yeast vectors expressing desaturase genes modified to contain these elements, (3) testing the vectors in a yeast expression system, (4) isolating regions from genomic DNA that are homologous to the beneficial cDNA elements, and (6) using them to construct chimeric or hybrid synthetic genes that produce functional and highly efficient desaturase activities in plant tissues.
Other features and advantages of the present invention will be better understood by reference to the drawings, detailed description and examples that follow.